The present invention relates to nucleic acid detection systems. More specifically, the invention relates to polynucleotide probes having binding specificity for rRNA or rDNA of bacteria that are members of the genus Staphylococcus.
Bacteria among the genus Staphylococcus are classified as members of the broad Bacillus-Lactobacillus-Streptococcus cluster. The closest phylogenetic relatives of the genus Staphylococcus include the genera Bacillus, Bronchothrix, Enterococcus, Listeria and Planococcus. Staphylococcal bacteria are non-motile, gram-positive cocci 30 having genomic molar percentages of guanine and cytosine (G+C) in the range of from to 39%. These bacteria commonly are found on skin and mucosal surfaces of humans. Notably, these organisms can become opportunistic pathogens following trauma to the skin. Indeed, S. aureus is frequently associated with infections of the skin. Infections of wounds and deep tissue with this Staphylococcal species can become life-threatening.
It is well established that two single strands of deoxyribonucleic acid (xe2x80x9cDNAxe2x80x9d) or ribonucleic acid (xe2x80x9cRNAxe2x80x9d) can associate or xe2x80x9chybridizexe2x80x9d with one another to form a double-stranded structure having two strands held together by hydrogen bonds between complementary base pairs. The individual strands of nucleic acid are formed from nucleotides that comprise the bases: adenine (A), cytosine (C), thymine (T), guanine (G), uracil (U) and inosine (I). In the double helical structure of nucleic acids, the base adenine hydrogen bonds with the base thymine or uracil, the base guanine hydrogen bonds with the base cytosine and the base inosine hydrogen bonds with adenine, cytosine or uracil. At any point along the chain, therefore, one may find the classical xe2x80x9cWatson-Crickxe2x80x9d base pairs A:T or A:U, T:A or U:A, and G:C or C:G. However, one may also find A:G, G:U and other xe2x80x9cwobblexe2x80x9d or mismatched base pairs in addition to the traditional (xe2x80x9ccanonicalxe2x80x9d) base pairs.
A double-stranded nucleic acid hybrid will result if a first single-stranded polynucleotide is contacted under hybridization-promoting conditions with a second single-stranded polynucleotide having a sufficient number of contiguous bases complementary to the sequence of the first polynucleotide. DNA/DNA, RNA/DNA or RNA/RNA hybrids may be formed under appropriate conditions.
Generally, a probe is a single-stranded polynucleotide having some degree of complementarity with the nucleic acid sequence that is to be detected (xe2x80x9ctarget sequencexe2x80x9d). Probes commonly are labeled with a detectable moiety such as a radioisotope, an antigen or a chemiluminescent moiety.
Descriptions of nucleic acid hybridization as a procedure for detecting particular nucleic acid sequences are given by Kohne in U.S. Pat. No. 4,851,330, and by Hogan et al., in U.S. Pat. Nos. 5,541,308 and 5,681,698. These references also describe methods for determining the presence of RNA-containing organisms in a sample which might contain such organisms. These procedures require probes that are sufficiently complementary to the ribosomal RNA (rRNA) of one or more non-viral organisms or groups of non-viral organisms. According to the method, nucleic acids from a sample to be tested and an appropriate probe are first mixed and then incubated under specified hybridization conditions. Conventionally, but not necessarily, the probe will be labeled with a detectable label. The resulting hybridization reaction is then assayed to detect and quantitate the amount of labeled probe that has formed duplex structures in order to detect the presence of rRNA contained in the test sample.
With the exception of viruses, all prokaryotic organisms contain rRNA genes encoding homologs of the procaryotic 5S, 16S and 23S rRNA molecules. In eucaryotes, these rRNA molecules are the 5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA which are substantially similar to the prokaryotic molecules. Probes for detecting specifically targeted rRNA subsequences in particular organisms or groups of organisms in a sample have been described previously. These highly specific probe sequences advantageously do not cross react with nucleic acids from any other bacterial species or infectious agent under appropriate stringency conditions.
The present invention provides polynucleotide probes that can be used to detect the members of the genus Staphylococcus in a highly specific manner.
One aspect of the present invention relates to an oligonucleotide probe that specifically hybridizes a Staphylococcal nucleic acid target region corresponding to E. coli 16S rRNA nucleotide positions 1276-1305 under a high stringency hybridization condition to form a detectable probe:target duplex. The oligonucleotide probe has a length of up to 100 nucleotides and includes at least 17 contiguous nucleotides contained within the sequence of SEQ ID NO:10. In a preferred embodiment, the oligonucleotide probe includes at least 30 contiguous nucleotides contained within the sequence of SEQ ID NO:10. The high stringency hybridization condition may be provided by either: (a) 0.48 M sodium phosphate buffer, 0. 1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, or (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. The oligonucleotide probe may be made of DNA, but also may include at least one nucleotide analog. For example, the nucleotide analog may include a methoxy group at the 2xe2x80x2 position of a ribose moiety. In one embodiment the invented oligonucleotide probe has the sequence of any one of SEQ ID NO:1 or the complement thereof, SEQ ID NO:2 or the complement thereof, and SEQ ID NO:3 or the complement thereof. In a preferred embodiment, the sequence of the oligonucleotide is given by SEQ ID NO:2 or SEQ ID NO:3, and the oligonucleotide is a helper oligonucleotide. Any of the disclosed oligonucleotides can include a detectable label. Particular examples of detectable labels include chemiluminescent labels and radiolabels. In another preferred embodiment, the oligonucleotide probe has a sequence given by SEQ ID NO:1, and further includes a detectable label. A highly preferred detectable label is an acridinium ester.
Another aspect of the present invention relates to a probe composition for detecting nucleic acids of bacteria that are members of the Staphylococcus genus. This composition includes an oligonucleotide probe that hybridizes under a high stringency condition to a Staphylococcal target region corresponding to E. coli 16S rRNA nucleotide positions 1276-1305 to form a detectable probe:target duplex. This oligonucleotide probe has a length of up to 100 nucleotide bases and includes at least 30 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. Under high stringency hybridization conditions the oligonucleotide probe specifically hybridizes nucleic acids present in Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus delphi, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus simulan and Staphylococcus warneri. In certain embodiments, the oligonucleotide probe is made of DNA. Exemplary high stringency hybridization conditions are provided by either: (a) 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, or (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 mM each of EDTA and EGTA. In a highly preferred embodiment, the oligonucleotide probe includes the sequence of SEQ ID NO:1 or the complement thereof. In another highly preferred embodiment, the length of the oligonucleotide probe is up to 60 bases. In and even more highly preferred embodiment of the invention, the oligonucleotide probe has the length and sequence of SEQ ID NO:1. Certain embodiments of the invented probe composition further include a detectable label on the oligonucleotide probe. For example, when the oligonucleotide probe has a length of up to 60 nucleotides, the probe may include a detectable label. Alternatively, when the probe has the length and sequence of SEQ ID NO:1 there can be included a detectable label. Regardless of whether the probe composition includes a labeled oligonucleotide probe of from 17-100 nucleotides in length, or from 17-60 nucleotides in length, or having the length and sequence of SEQ ID NO:1 the detectable label may be a chemiluminescent label, such as an acridinium ester, or a radiolabel. It is preferred that the invented probe composition include at least one helper oligonucleotide that facilitates formation of the detectable probe:target duplex under high stringency hybridization conditions. These helper oligonucleotides may include at least one nucleotide analog, such as a ribose moiety having a methoxy group disposed at the 2xe2x80x2 position. In a highly preferred embodiment of the invented probe composition, the helper oligonucleotide has a sequence given by SEQ ID NO:2 or SEQ ID NO:3.
Yet another aspect of the invention relates to a method for detecting the presence of Staphylococcus bacteria in a test sample. This method involves steps for providing to the test sample a probe composition that includes an oligonucleotide probe that hybridizes under a high stringency condition to a Staphylococcal target region corresponding to E. coli 16S rRNA nucleotide positions 1276-1305 to form a detectable probe:target duplex. The oligonucleotide probe has a length of up to 100 nucleotide bases and includes at least 17, or more preferably at least 30 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. Under high stringency hybridization conditions the oligonucleotide probe specifically hybridizes nucleic acids present in Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus delphi, Staphylococcus epidermis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus simulan and Staphylococcus warneri. Thereafter, the resulting mixture is hybridized under high stringency conditions so that any nucleic acids from Staphylococcus bacteria that may be present in the test sample form probe:target duplexs with the probe oligonucleotide. Finally, the method involves detecting the probe:target duplexs as an indicator of the presence of Staphylococcus bacteria in the test sample. In one embodiment of the invented method the test sample includes bacteria, and there is conducted a preliminary step for releasing nucleic acids from any bacteria that may be present in said test sample. In a different embodiment of the method the test sample is a lysate. In general, high stringency hybridization conditions can be provided by either: (a) 0.48 M sodium phosphate buffer, 0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, or (b) 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10 nM each of EDTA and EGTA. However, it is to be understood that other high stringency hybridization conditions can give good results. In a preferred embodiment, the oligonucleotide probe has the length and sequence of SEQ ID NO:1, and optionally may include a detectable label. This detectable label may be an acridinium ester. When this is the case the detecting step in the invented method may include a step for performing luminometry to detect any of the probe:target duplexs that are formed during the hybridization step. When the oligonucleotide probe has the length and sequence of SEQ ID NO:1, the probe composition may further include at least one helper oligonucleotide that facilitates formation of the probe:target duplex. Highly preferred helper oligonucleotides have the sequences of SEQ ID NO:2 and SEQ ID NO:3.
Still yet another aspect of the invention relates to a kit that can be used for detecting the presence in a test sample of nucleic acids from bacteria that are members of the Staphylococcus genus. The kit contains a probe composition that includes an oligonucleotide probe that hybridizes under a high stringency condition to a Staphylococcal target region corresponding to E. coli 16S rRNA nucleotide positions 1276-1305 to form a detectable probe:target duplex. The oligonucleotide probe has a length of up to 100 nucleotide bases and includes at least 30 contiguous nucleotides contained within the sequence of SEQ ID NO:10 or the complement thereof. Under high stringency hybridization conditions the oligonucleotide probe specifically hybridizes nucleic acids present in Staphylococcus aureus, Staphylococcus cohnii, Staphylococcus delphi, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus simulan and Staphylococcus warneri. Also included in the kit are printed instructions specifying, in order of implementation, the steps to be followed for detecting nucleic acids from bacteria that are members of the Staphylococcus genus by detecting a complex between the oligonucleotide probe and a Staphylococcus nucleic acid target. Both the probe composition and the printed instructions are in packaged combination with each other.
Definitions
As used herein, the following terms have the given meanings unless expressly stated to the contrary.
A xe2x80x9cnucleotidexe2x80x9d is a subunit of a nucleic acid consisting of a phosphate group, a 5-carbon sugar and a nitrogenous base. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is 2xe2x80x2-deoxyribose. For a 5xe2x80x2-nucleotide, the sugar contains a hydroxyl group (xe2x80x94OH) at the 5xe2x80x2-carbon-5. The term also includes analogs of such subunits, such as a methoxy group at the 2xe2x80x2 position of the ribose (OMe). As used herein, methoxy oligonucleotides containing xe2x80x9cTxe2x80x9d residues have a methoxy group at the 2xe2x80x2 position of the ribose moiety, and a uracil at the base position of the nucleotide.
A xe2x80x9cnon-nucleotide unitxe2x80x9d is a unit which does not significantly participate in hybridization of a polymer. Such units must not, for example, participate in any significant hydrogen bonding with a nucleotide, and would exclude units having as a component one of the five nucleotide bases or analogs thereof.
An xe2x80x9coligonucleotidexe2x80x9d is a nucleotide polymer having two or more nucleotide subunits covalently joined together. Oligonucleotides are generally about 10 to about 100 nucleotides in length. The sugar groups of the nucleotide subunits may be ribose, deoxyribose, or modified derivatives thereof such as OMe. The nucleotide subunits may by joined by linkages such as phosphodiester linkages, modified linkages or by non-nucleotide moieties that do not prevent hybridization of the oligonucleotide to its complementary target nucleotide sequence. Modified linkages include those in which a standard phosphodiester linkage is replaced with a different linkage, such as a phosphorothioate linkage, a methylphosphonate linkage, or a neutral peptide linkage.
Nitrogenous base analogs also may be components of oligonucleotides in accordance with the invention.
A xe2x80x9ctarget nucleic acidxe2x80x9d is a nucleic acid comprising a target nucleic acid sequence.
A xe2x80x9ctarget nucleic acid sequence,xe2x80x9d xe2x80x9ctarget nucleotide sequencexe2x80x9d or xe2x80x9ctarget sequencexe2x80x9d is a specific deoxyribonucleotide or ribonucleotide sequence that can be hybridized by an oligonucleotide.
An xe2x80x9coligonucleotide probexe2x80x9d is an oligonucleotide having a nucleotide sequence sufficiently complementary to its target nucleic acid sequence to be able to form a detectable hybrid probe:target duplex under high stringency hybridization conditions. An oligonucleotide probe is an isolated chemical species and may include additional nucleotides outside of the targeted region as long as such nucleotides do not prevent hybridization under high stringency hybridization conditions. Non-complementary sequences, such as promotor sequences, restriction endonuclease recognition sites, or sequences that confer a desired secondary or tertiary structure such as a catalytic active site can be used to facilitate detection using the invented probes. An oligonucleotide probe optionally may be labeled with a detectable moiety such as a radioisotope, a fluorescent moiety, a chemiluminescent moiety, an enzyme or a ligand, which can be used to detect or confirm probe hybridization to its target sequence. Oligonucleotide probes are preferred to be in the size range of from 10 to 100 nucleotides in length.
A xe2x80x9cdetectable moietyxe2x80x9d is a molecule attached to, or synthesized as part of, a nucleic acid probe. This molecule should be uniquely detectable and will allow the probe to be detected as a result. These detectable moieties are often radioisotopes, chemiluminescent molecules, enzymes, haptens, or even unique oligonucleotide sequences.
A xe2x80x9chybridxe2x80x9d or a xe2x80x9cduplexxe2x80x9d is a complex formed between two single-stranded nucleic acid sequences by Watson-Crick base pairings or non-canonical base pairings between the complementary bases.
xe2x80x9cHybridizationxe2x80x9d is the process by which two complementary strands of nucleic acid combine to form a double-stranded structure (xe2x80x9chybridxe2x80x9d or xe2x80x9cduplexxe2x80x9d).
xe2x80x9cComplementarityxe2x80x9d is a property conferred by the base sequence of a single strand of DNA or RNA which may form a hybrid or double-stranded DNA:DNA, RNA:RNA or DNA:RNA through hydrogen bonding between Watson-Crick base pairs on the respective strands. Adenine (A) ordinarily complements thymine (T) or uracil (U), while guanine (G) ordinarily complements cytosine (C).
xe2x80x9cMismatchxe2x80x9d refers to any pairing, in a hybrid, of two nucleotides which do not form canonical Watson-Crick hydrogen bonds. In addition, for the purposes of the following discussions, a mismatch can include an insertion or deletion in one strand of the hybrid which results in an unpaired nucleotide(s).
The term xe2x80x9cstringencyxe2x80x9d is used to describe the temperature and solvent composition existing during hybridization and the subsequent processing steps. Under high stringency conditions only highly complementary nucleic acid hybrids will form; hybrids without a sufficient degree of complementarity will not form. Accordingly, the stringency of the assay conditions determines the amount of complementarity needed between two nucleic acid strands forming a hybrid. Stringency conditions are chosen to maximize the difference in stability between the hybrid formed with the target and the non-target nucleic acid. Exemplary high stringency conditions are provided in the working Examples.
The term xe2x80x9cprobe specificityxe2x80x9d refers to a characteristic of a probe which describes its ability to distinguish between target and non-target sequences.
The term xe2x80x9cvariable regionxe2x80x9d refers to a nucleotide polymer which differs by at least one base between the target organism and non-target organisms contained in a sample.
A xe2x80x9cconserved regionxe2x80x9d is a nucleic acid subsequence which is not variable between at least two different polynucleotides.
xe2x80x9cBacteriaxe2x80x9d are members of the phylogenetic group eubacteria, which is considered one of the three primary kingdoms.
The term xe2x80x9csequence divergencexe2x80x9d refers to a process by which nucleotide polymers become less similar during evolution.
The term xe2x80x9csequence convergencexe2x80x9d refers to a process by which nucleotide polymers become more similar during evolution.
xe2x80x9cTmxe2x80x9d refers to the temperature at which 50% of the probe is converted from the hybridized to the unhybridized form.
A xe2x80x9chelper oligonucleotidexe2x80x9d is an oligonucleotide that binds a region of a target nucleic acid other than the region that is bound by an oligonucleotide probe. Helper oligonucleotides impose new secondary and tertiary structures on the targeted region of the single-stranded nucleic acid so that the rate of binding of the oligonucleotide probe is accelerated. Although helper oligonucleotides are not labeled with a detectable label when used in conjunction with labeled oligonucleotide probes, they facilitate binding of labeled probes and so indirectly enhance hybridization signals.
The phrases xe2x80x9cconsist essentially ofxe2x80x9d or xe2x80x9cconsisting essentially ofxe2x80x9d means that the oligonucleotide has a nucleotide sequence substantially similar to a specified nucleotide sequence. Any additions or deletions are non-material variations of the specified nucleotide sequence which do not prevent the oligonucleotide from having its claimed property, such as being able to preferentially hybridize under high stringency hybridization conditions to its target nucleic acid over non-target nucleic acids.
One skilled in the art will understand that substantially corresponding probes of the invention can vary from the referred-to sequence and still hybridize to the same target nucleic acid sequence. This variation from the nucleic acid may be stated in terms of a percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe and its target sequence. Probes of the present invention substantially correspond to a nucleic acid sequence if these percentages are from 100% to 80% or from 0 base mismatches in a 10 nucleotide target sequence to 2 bases mismatched in a 10 nucleotide target sequence. In preferred embodiments, the percentage is from 100% to 85%. In more preferred embodiments this percentage is from 90% to 100%; in other preferred embodiments, this percentage is from 95% to 100%.
By xe2x80x9csufficiently complementaryxe2x80x9d or xe2x80x9csubstantially complementaryxe2x80x9d is meant nucleic acids having a sufficient amount of contiguous complementary nucleotides to form, under high stringency hybridization conditions, a hybrid that is stable for detection.
By xe2x80x9cnucleic acid hybridxe2x80x9d or xe2x80x9cprobe:target duplexxe2x80x9d is meant a structure that is a double-stranded, hydrogen-bonded structure, preferably 10 to 100 nucleotides in length, more preferably 14 to 50 nucleotides in length. The structure is sufficiently stable to be detected by means such as chemiluminescent or fluorescent light detection, autoradiography, electrochemical analysis or gel electrophoresis. Such hybrids include RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.
By xe2x80x9cnegative sensexe2x80x9d is meant a nucleic acid molecule perfectly complementary to a reference (i.e., sense) nucleic acid molecule.
xe2x80x9cRNA and DNA equivalentsxe2x80x9d refer to RNA and DNA molecules having the same complementary base pair hybridization properties. RNA and DNA equivalents have different sugar groups (i.e., ribose versus deoxyribose), and may differ by the presence of uracil in RNA and thymine in DNA. The difference between RNA and DNA equivalents do not contribute to differences in substantially corresponding nucleic acid sequences because the equivalents have the same degree of complementarity to a particular sequence.
By xe2x80x9cpreferentially hybridizexe2x80x9d is meant that under high stringency hybridization conditions oligonucleotide probes can hybridize their target nucleic acids to form stable probe:target hybrids (thereby indicating the presence of the target nucleic acids) without forming stable probe:non-target hybrids (that would indicate the presence of non-target nucleic acids from other organisms). Thus, the probe hybridizes to target nucleic acid to a sufficiently greater extent than to non-target nucleic acid to enable one skilled in the art to accurately detect the presence of bacteria in the Staphylococcus genus and distinguish their presence from that of other organisms. Preferential hybridization can be measured using techniques known in the art and described herein. For example, when compared with hybridization to C. albicans nucleic acids, oligonucleotide probes of the invention preferentially hybridize nucleic acids from bacteria in the Staphylococcus genus by about 500-3,000 fold.
A xe2x80x9ctarget nucleic acid sequence regionxe2x80x9d of bacteria in the Staphylococcus genus refers to a nucleic acid sequence present in nucleic acid or a sequence complementary thereto found in Staphylococcal bacteria, which is not present in nucleic acids of other species. Nucleic acids having nucleotide sequences complementary to a target sequence may be generated by target amplification techniques such as polymerase chain reaction (PCR) or transcription mediated amplification (e.g., Kacian and Fultz, Nucleic Acid Sequence Amplification Methods, U.S. Pat. No. 5,824,518).